ABSTRACT Urinary tract infections (UTIs), including pyelonephritis, are among the most common and serious infections encountered in clinical practice. No proven treatment options exist to prevent UTI. New strategies are needed to augment the ability of host defenses to prevent UTI and minimize UTI-associated morbidity. A growing body of evidence, from our laboratory and others suggests that antimicrobial peptides (AMP), an essential component of the innate immune response, protect the urinary tract from invasive bacterial infection. Our research team has identified Ribonuclease 7 (RNase 7) as a potent and highly abundant human AMP that shields the urothelium from uropathogenic E. coli (UPEC). Our published data suggest that RNase 7 is an ideal AMP to develop as a UTI therapeutic because: (A) it has potent antibacterial activity; (B) it is highly abundant in the urinary tract; (C) it is produced by cell types that are targeted by UPEC; and (D) it has minimal toxicity. Our emerging data suggest that RNase 7 induction shields the urothelium from UPEC, while suppressed RNase 7 production renders it susceptible to pathogens. Together, these findings provide strong support to our central hypothesis that RNase 7 is biologically necessary to maintain urine sterility and prevent UTI. Our current understanding of RNase 7's effects on innate defenses is limited in vivo because its expression is absent in the laboratory mouse and restricted to higher order vertebrates and humans. To fill this key knowledge gap, we developed two novel humanized RNase 7 mouse models. These models will be used to complete our overall objective of this application, which is to further investigate the essential contributions of RNase 7 to urine sterility. To test our central hypothesis, we will evaluate how cell-specific RNase 7 expression impacts UTI risk in vivo using a novel Rosa26 knock-in mouse (Aim 1). In Aim 2, we will investigate the molecular mechanisms that regulate RNase 7 expression using a novel humanized transgenic mouse that expresses RNase 7 under the control of its own promoter. In Aim 3, we will evaluate how human genetic polymorphisms affect RNase 7 expression and antimicrobial activity. Completion of the proposed Aims will further define the necessary contributions of RNase 7 to urine sterility and may provide the foundation to develop RNase 7 as a novel therapeutic that improves UTI outcomes. Given the clinical impact of UTI, in an era of emerging antibiotic resistant uropathogens, identifying mechanisms to develop RNase 7 as a new UTI therapy may have significant benefits to public health.